Novel reporters of mitochondria-associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis
By Shohei Sakai, Seiji Watanabe, Okiru Komine, Akira Sobue, and Koji Yamanaka
Excerpt from the article published in The FASEB Journal. 2021; 35:e21688. pPublished: 18 June 2021, DOI: https://doi.org/10.1096/fj.202100137R
Editor’s Highlights
- Aberrant regulation of MAM is associated with various neurological diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that selectively affects motor neurons.
- MAM disruption is a key pathomechanism in familial ALS and in sporadic ALS.
- Sigma-1 receptor (Sig1R), a gene product of SIGMAR1, is a MAM-specific chaperone protein, and the loss of Sig1R function is responsible for juvenile ALS.
- Pharmacological activation of Sig1R extended the survival of SOD1-linked ALS (ALS1)
- SIGMAR1 has a protective role in the maintenance of MAM integrity.
Abstract
The mitochondria-associated membrane (MAM) is a functional subdomain of the endoplasmic reticulum membrane that tethers to the mitochondrial outer membrane and is essential for cellular homeostasis. A defect in MAM is involved in various neurological diseases, including amyotrophic lateral sclerosis (ALS). Recently, we and others reported that MAM was disrupted in the models expressing several ALS-linked genes, including SOD1, SIGMAR1, VAPB, TARDBP, and FUS, suggesting that MAM disruption is deeply involved in the pathomechanism of ALS. However, it is still uncertain whether MAM disruption is a common pathology in ALS, mainly due to the absence of a simple, quantitative tool for monitoring the status of MAM. In this study, to examine the effects of various ALS-causative genes on MAM, we created the following two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. The MAMtrackers could detect MAM disruption caused by suppression of SIGMAR1 or the overexpression of ALS-linked mutant SOD1 in living cells. Moreover, the MAMtrackers have an advantage in their ability to monitor reversible changes in the MAM status induced by nutritional conditions. We used the MAMtrackers with an expression plasmid library of ALS-causative genes and noted that 76% (16/21) of the genes altered MAM integrity. Our results suggest that MAM disruption is a common pathological feature in ALS. Furthermore, we anticipate our MAMtrackers, which are suitable for high-throughput assays, to be valuable tools to understand MAM dynamics.
1 INTRODUCTION
The mitochondria-associated membrane (MAM) is a functional subdomain of the endoplasmic reticulum (ER) membrane that physically contacts the mitochondrial outer membrane (MOM). Aberrant regulation of MAM is associated with various neurological diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that selectively affects motor neurons. Although most ALS cases are sporadic, approximately 10% are inherited, and over 20 causative genes for inherited ALS have currently been identified.1 Previous studies based on the analysis of various ALS-causative genes have identified several pathogenic mechanisms for ALS, including RNA metabolism disturbance, abnormal proteostasis, altered axonal transport, neuroinflammation, and dysfunction of organelles such as the ER and mitochondria.2
We recently reported MAM disruption as a common pathomechanism in SOD1– and SIGMAR1-linked ALS.3 Sigma-1 receptor (Sig1R), a gene product of SIGMAR1, is a MAM-specific chaperone protein,4 and the loss of Sig1R function is responsible for juvenile ALS (ALS16).5Alternatively, ALS-linked mutants of Cu/Zn superoxide dismutase (SOD1) have high propensities for aggregation and accumulate at MAM, leading to MAM disruption.3 The significance of MAM integrity in neurodegeneration was also suggested in studies reporting that pharmacological activation of Sig1R extended the survival of SOD1-linked ALS (ALS1) model mice.6, 7 Moreover, an association between neurodegeneration and MAM integrity is reported in multiple ALS-causative genes: VAPB, TARDBP, and FUS.8–11 These findings suggest that defects in MAM integrity are deeply involved in the pathomechanism of ALS. Although over 20 causative genes for inherited ALS have been identified, it remains uncertain whether all the ALS-causative genes compromise MAM integrity and how MAM disruption is involved in neurodegeneration.
The absence of a simple, quantitative, and high-throughput detection system makes it challenging to monitor the MAM status. To date, MAM has been analyzed by electron microscopy (EM), fluorescent microscopy, proximity ligation assay (PLA), and bimolecular fluorescence complementation (BiFC). EM is a powerful tool to visualize the MAM ultrastructure directly. However, it requires a considerable amount of time and effort and is inapplicable to living cells. Fluorescent microscopy is an alternate, time-saving approach that is applicable to living cells by indirect observation of MAM labeled with fluorescent probes. However, due to its relatively low resolution (200-300 nm),12 its suitability for evaluating MAM is controversial. Although PLA is a more accurate method to detect protein-protein interactions (<40 nm),13 it requires fixation and highly specific antibodies for proper evaluation. Recently, some MAM reporters were developed based on the split green fluorescent protein (split GFP), a well-used molecule in the BiFC technology.14–16 These MAM reporters appear to be attractive tools that overcome the limitation of resolution and can be used to evaluate living cells. However, the irreversible interaction of the two components of split GFP may introduce an artifact of the dynamic structure of MAM. Thus, this disadvantage of split GFP cannot be ignored. As mentioned above, each conventional method has its advantages and disadvantages. However, they share the weakness of limited performance for high-throughput quantification of the MAM status. Therefore, we aimed to establish a new tool suitable for high-throughput screening and quantification of MAM in living cells.
In this study, we created two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. These MAMtrackers were more suitable for monitoring reversible changes in the MAM status than a split GFP-based reporter. Moreover, we performed a screening for MAM disruption by using the MAMtrackers and a mammalian ALS-causative gene expression library and found that 76% (16/21) of the examined ALS-causative genes altered MAM integrity. Our results suggest that MAM disruption is a common hallmark in ALS and that our novel MAM reporters, MAMtrackers, are valuable tools to monitor MAM dynamics in living cells.
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3 RESULTS
3.1 Development of two novel MAM reporters, MAMtracker-Luc and MAMtracker-Green
To enable high-throughput screening and quantification of MAMs, we developed two novel MAM reporters, MAMtracker-Luc and MAMtracker-Green (Figure 1A,B). MAMtracker-Luc is a luminescence-based reporter using the NanoLuc Binary Technology (NanoBiT) system.24, 25In the NanoBiT system, NanoLuc, a luciferase derived from Oplophorus gracilirostris,24consists of a large NanoBiT fragment (LgBiT; 18 kDa) and a small NanoBiT fragment (SmBiT; 1.3 kDa).25 We fused an ER anchor sequence to the C-terminus of LgBiT and human TOM20 to N-terminus of SmBiT to utilize NanoLuc as a MAM reporter, allowing to emit bioluminescence when the ER and mitochondria were in close proximity (Figure 1A). Alternatively, MAMtracker-Green was based on dimerization-dependent GFP (ddGFP), which emits fluorescence only when ddGFP-A and ddGFP-B form a heterodimer.26 As with MAMtracker-Luc, each component of MAMtracker-Green was fused to an ER anchor sequence or a mitochondria-targeting signal (mito signal) (Figure 1B). The ER anchor sequence of both the MAMtrackers and the mitochondria-targeting sequence of MAMtracker-Green is based on the work by Csordás and colleagues.27 Unlike the previous studies,25, 26 we conjugated the two parts of the MAMtrackers in one vector using a self-cleaving P2A peptide for the bicistronic expression of each component. This strategy enabled us to perform experiments with a single transfection procedure and ensured an equal expression level of the two components. We confirmed each MAMtracker component was successfully separated in the N2a cells by immunoblotting (Figure 1C,D).
Strategy to detect and quantify MAMs using two novel MAM reporters: MAMtracker-Luc and MAMtracker-Green. A, B, Schematic illustration of the split NanoLuc-based MAM reporter, MAMtracker-Luc (A), and the ddGFP-based MAM reporter, MAMtracker-Green (B). C, D, Representative immunoblots for each component of MAMtracker-Luc (C) and MAMtracker-Green (D). The lysates of N2a cells stably expressing MAMtracker-Luc (N2a･MAMLuc) (C) and N2a cells transiently transfected with MAMtracker-Green (D) were analyzed. Black and white arrowheads indicate self-cleaved and uncleaved proteins, respectively. Note that almost all proteins were successfully cleaved into the two self-cleaved components as designed. An asterisk indicates non-specific bands
3.2 MAMtrackers specifically localize to MAM
Prior to the use of the MAMtrackers, we confirmed that all the MAMtracker components had successfully localized to the intended subcellular fractions (Figure 2A,B). It should be noted that ER-anchored LgBiT and ddGFP-A were mainly fractionated into the MAM fraction and not P3, which predominantly contains smooth ER and microsomes. The appropriate localization of the MAMtracker components was also confirmed by immunocytochemistry. SmBiT and ddGFP-B were colocalized with the mitochondria-specific proteins, superoxide dismutase 2 (SOD2) and TOM20, respectively (Figure 2C,D, upper panels), whereas LgBiT and ddGFP-A were colocalized with Sig1R, a MAM-specific protein (Figure 2C,D, lower panels). Moreover, MAMtracker-Green fluorescence was detected at the points where the signals of MitoTracker and ER-Tracker were overlapped (Figure 2E,F). Of note, MAMtracker-Green did not cause apparent morphological changes in the mitochondria and the ER, and both the MAMtrackers did not induce any cytotoxicities (Figure S1A,B). Collectively, these data indicate that both the MAMtrackers specifically localize to MAM without affecting the physiological state of MAM.
Subcellular localization of MAMtrackers. A, B, Subcellular localization of each component of MAMtracker-Luc (A) and MAMtracker-Green (B) was determined by subcellular fractionation followed by immunoblotting. N2a･MAMLuc cells (A) and N2a cells transiently transfected with MAMtracker-Green (B) were fractionated, and each fraction was confirmed by the following fraction-specific marker proteins as indicated: Histone H2A, P1 marker; voltage-dependent anion channel (VDAC), mitochondrial marker; protein disulfide isomerase (PDI), P3 and MAM marker; Sig1R, MAM marker; and SOD1, cytosolic marker. C, D, Subcellular localization of MAMtracker-Luc (C) and MAMtracker-Green (D) was determined by immunocytochemistry in N2a cells. Human TOM20-SmBiT and LgBiT-ER anchors were immunostained with a human TOM20-specific antibody and a NanoLuc antibody, respectively. Mitochondrial SOD2 and TOM20 were stained as mitochondrial markers. Sig1R was stained as a MAM marker. E, The fluorescence signal of MAMtracker-Green was specifically observed at MAMs in living HeLa cells. Representative confocal images of HeLa cells transiently transfected with MAMtracker-Green were stained with MitoTracker Red CMX ROS and ER-Tracker Blue-White DPX. F, Magnified confocal images of the area indicated by the square in (E) (upper panels) and relative fluorescence intensity of each channel at the points along the arrow (lower graph). Scale bars: 10 µm
3.3 MAMtrackers are useful tools to monitor the MAM status in living cells
To confirm that MAMtrackers are useful to monitor the MAM status, we investigated whether the MAMtrackers could detect intracellular MAM upregulation under starvation14, 16, 28 and MAM downregulation caused by the elimination of Sig1R or the overexpression of mutant SOD1.3 Increases in the luminescence of MAMtracker-Luc were dependent on the amounts of the transfected plasmid and duration of starvation (Figure 3A). Conversely, the luminescence of MAMtracker-Luc was decreased by the elimination of Sig1R using siRNA or the overexpression of SOD1G85R (Figure 3B). MAMtracker-Green was validated using the same conditions as MAMtracker-Luc. As with MAMtracker-Luc, the fluorescent signal of MAMtracker-Green was increased by starvation (Figures 3C and S2A) and decreased by Sig1R elimination (Figures 3D and S2B) or SODG85R overexpression (Figures 3E and S2C). Notably, aggregated SODG85R induced morphological abnormalities of MAM labeled with MAMtracker-Green (Figure 3E, arrow). These findings were consistent with our previous data that aggregated mutant SOD1 is prone to accumulate at MAM and consequently disrupts MAM.3
Quantification of MAM alterations in living cells using MAMtrackers. A, Quantification of MAM in N2a cells under starvation using MAMtracker-Luc. N2a cells were transiently transfected with MAMtracker-Luc, and the luminescence was quantified with or without a substrate for NanoLuc. For the starvation condition, cells were incubated with Hank’s buffered saline solution (HBSS) for 4 hours just before the luminescence measurement. The raw data were normalized using a control sample (blue bar), and the relative values are plotted as the mean ± standard error of the mean (SEM). Two independent experiments were performed in duplicate. B, Quantification of MAM with Sig1R suppression or mutant SOD1 overexpression using N2a･MAMtracker-Luc cells. The luminescence of differentiated N2a･MAMLuc stable cells transiently transfected with the indicated siRNAs or expression plasmids was measured. The luminescence relative to the control (black bar) was plotted as the mean ± SEM. Three independent experiments were performed in triplicate. C, MAM increase in starved N2a cells was visualized using MAMtracker-Green. Representative confocal images of N2a cells transiently transfected with MAMtracker-Green are shown (left panels). The starved cells were incubated with HBSS for 4 hours just prior to the imaging analysis. The cells were counterstained with Hoechst. Relative fluorescence intensities normalized by the fed control sample are shown in a dot plot (right panel). In each sample, 60 cells from three independent experiments were analyzed. D, E, MAM reduction in N2a cells with Sig1R suppression (D) or mutant SOD1 overexpression (E) visualized using MAMtracker-Green. Representative confocal images of differentiated N2a cells transiently co-transfected with MAMtracker-Green and the indicated siRNAs or expression plasmids (left panels). The cells in (D) were counterstained with Hoechst. For clarity, the mCherry image of SODG85R in (E) was captured with a higher optical gain than that of the WT. Relative fluorescence intensities were normalized using the control samples (D; siCtrl, E; SOD1WT) and are shown in dot plots (right panel). In each sample, 75 cells from three independent experiments were analyzed. Accumulation of aggregated mutant SOD1 at MAM (E, arrow). F-H, Detection of MAM using the MAMtrackers in primary cultured astrocytes (F, G) or mouse embryonic fibroblasts (MEFs) (H). Primary cultured astrocytes of wild-type C57BL/6J mice were transfected with MAMtracker-Green (F) or -Luc (G). The cells were treated with growth media (Fed) or HBSS (Starved) for 4 hours prior to the measurement (G). Primary MEFs were also established from wild-type or Sigmar1 knock-out (Sig1R-KO) C57/BL6J mice and transfected with MAMtracker-Luc (H). Note that both MAM induction under starvation and MAM reduction by Sig1R deficiency were successfully observed as in the cultured cell lines. One way-ANOVA with Tukey’s multiple comparison test was performed for group comparisons (A and B). Unpaired t-test with Welch’s correction was performed for single comparisons (C–E, G and H). *P < .05, **P < .01, ***P < .001, and ****P < .0001. Scale bars: 10 µm
Although confocal imaging of ER and mitochondria labeled with fluorescent probes is a well-used and straightforward method, its application has been limited to large and flat cell lines such as U2OS,29 CV-1,8, 10 and HeLa.30 This is probably because such morphological features help the confocal microscope to capture the microstructure at a limited resolution. Indeed, we confirmed that immunofluorescent staining of ER-mitochondria tethering sites was able to detect MAM upregulation in HeLa cells (Figure S3A), but in N2a, a small and round neuronal cell line, we failed to obtain proper images with sufficient resolution to perform colocalization analyses (Figure S3B). In contrast, the MAMtrackers successfully detect MAM changes in N2a cells (Figure 3A-E). Moreover, we confirmed that the MAMtrackers are available to monitor the MAM status in primary cultured cells. Using MAMtracker-Green and -Luc, we successfully visualized MAM in primary cultured astrocytes (Figure 3F) and detected MAM upregulation under starvation (Figure 3G). MAM downregulation in MEFs derived from Sig1R knockout mice was also detectable using MAMtracker-Luc (Figure 3H). All these data indicate that the MAMtrackers are valuable tools to monitor the status of MAM in various types of cells.
3.4 MAMtrackers have advantages in detecting reversible changes in MAM dynamics in living cells
Recently, split GFP was applied to detect organelle contact sites, including MAM.14–16Because the interaction of each MAMtracker component is reversible due to its relatively high dissociation constant (Kd = 9 µM for MAMtracker-Green and Kd = 190 µM for MAMtracker-Luc),25, 26 it is expected that the MAMtrackers will be more suitable for detecting reversible changes in MAM than the BiFC-based MAM detection tools. To compare the MAMtrackers with the BiFC-based system, we generated a construct to express split GFP anchored at MAM in the same manner as the MAMtrackers (MAM-split GFP) (Figures 4A,B, and S4). Although both MAM-split GFP and MAMtracker-Green were able to detect an increase of MAM under starvation, MAMtracker-Green was more sensitive for detecting recovery from starvation (Figures 4C,D). The fluorescence of MAMtracker-Green returned to the basal level after 8 hours of the recovery, whereas the fluorescence of MAM-split GFP remained at a significantly higher level than baseline even after 12 hours of the recovery. Similar to MAMtracker-Green, the luminescence of MAMtracker-Luc was also returned to the basal level within 12 hours of the recovery (Figure 4E). These data indicate that the MAMtrackers are reversible MAM reporters and suitable to detect the intracellular MAM dynamics in living cells.
MAMtrackers have advantages in detecting reversible changes in MAM dynamics. A, Schematic illustration of the split GFP-based MAM reporter, MAM-split GFP. B, Representative immunoblots for each component of MAM-split GFP. The lysates of N2a cells transiently transfected with pMAM-split GFP were analyzed by immunoblotting for Myc (GFP11) and HA (GFP1-10). Black and white arrowheads indicate self-cleaved and uncleaved proteins, respectively. Note that almost all the proteins were successfully cleaved into the two self-cleaved components as designed. C, D, MAMtracker-Green is more sensitive to detect recovery from starvation than MAM-split GFP. N2a cells transiently expressing MAM-split GFP or MAMtracker-Green were starved in HBSS, then recovered in growth media for the indicated time. Representative confocal images of the cells counterstained with Hoechst are shown (C). Relative fluorescence intensities normalized by the fed control samples are shown as the mean ± SEM (D). In each sample, 60 cells from three independent experiments were quantified. E, Quantification of MAM reduction during recovery from starvation in N2a･MAMLuc stable cells. The cells were starved in HBSS for 4 hours, and then recovered in growth media for the indicated times before measuring the luminescence. Relative luminescence as normalized by the fed control sample is shown as the mean ± SEM. Three independent experiments were performed in duplicate. One way-ANOVA with Dunnett’s multiple comparison test was performed for group comparisons. *P < .05 and ****P < .0001. ns, not significant vs. the fed controls. Scale bar: 10 µm
3.5 MAM disruption is a common pathological feature in various causes of inherited ALS
Finally, we screened for MAM disruptions using MAMtracker-Luc (N2a･MAMLuc stable cells) and a mammalian gene expression library of ALS-causative genes.18 The screening results are shown in Figure 5A-D. We found that the amounts of MAM were altered by the expression of 14 ALS-causative genes in this screening. Including the results of SIGMAR1 and SOD1 (Figure 3D,E), our screening showed various ALS-causative genes (16/21 genes, 76%) affected MAM, and interestingly, the vast majority of those genes (14/16 genes) caused MAM disruption. In contrast, wild-type fused in sarcoma (FUS) and TANK-binding kinase 1 (TBK1) increased the amount of MAM. This finding is consistent with the hypothesis that a loss-of-function mechanism is involved in FUS- and TBK1-linked ALS.31–35 Moreover, we revalidated the representative MAMtracker-Luc screening results using MAMtracker-Green (Figure 5E,F). Consistent with the results using MAMtracker-Luc, wild-type FUS increased the fluorescence of MAMtracker-Green, and ALS-linked mutant of valosin-containing protein (VCP), VCPA232E, decreased the fluorescence. Collectively, our findings suggest that disruption of MAM is a common pathological feature in ALS, at least in inherited ALS cases.
Disruption of MAM integrity is a common pathological feature in cells expressing ALS-causative genes. A–D, Impairment of MAM was induced in N2a･MAMLuc stable cells expressing various ALS-causative genes and their mutants. The luminescence of differentiated N2a･MAMLuc cells transfected with the indicated ALS-causative genes was measured. The raw luminescence values were compensated by the cell viability measured by an MTS assay. The relative luminescent values normalized by the mCherry-transfected control are shown as the mean ± SEM. In each sample, three independent experiments were performed in triplicate. The ALS-causative genes were classified into four groups according to their putative functions: DNA/RNA binding (A), trafficking (B), protein degradation (C), and cytoskeleton/others (D). E, F, Changes in MAM induced by the expression of WT and mutant FUS or VCP visualized using MAMtracker-Green. Representative confocal images of N2a cells transiently co-transfected with MAMtracker-Green and FUS or VCP (E). Relative fluorescence intensities normalized by the mCherry-transfected control are shown in a dot plot (F). In each sample, 80 cells from three independent experiments were quantified. One way-ANOVA with Dunnett’s multiple comparison test was performed for group comparisons. *P < .05. ns, not significant vs. the mCherry controls. Scale bar: 10 µm
4 DISCUSSION
One of the main goals of this study was to clarify whether all the ALS-causative genes affected MAM integrity in living cells. To this end, we created two novel MAM reporters, MAMtracker-Luc and MAMtracker-Green based on NanoBiT and ddGFP, respectively. We demonstrated that both the MAMtrackers could function as quantitative and reversible MAM reporters in living cells. Using the MAMtrackers, we performed a comprehensive screening of ALS-causative genes to determine their impact on MAM; our results revealed that not all but 76% (16/21) of the examined ALS-causative genes altered MAM integrity and the vast majority of them impaired it. These results indicate that disruption of MAM is a common hallmark in various types of ALS.
To date, various tools and approaches have been developed to evaluate MAM. Among them, the simplest one is probably confocal microscopy imaging of ER- and mitochondria-targeted fluorescent probes. This method has been widely used in both living and fixed cells, but its application has been limited to large and flat cell lines. In contrast, we demonstrated that the MAMtrackers are applicable to N2a, a small and round cell line that is not suitable for MAM evaluation using the conventional confocal microscopy. Moreover, the MAMtrackers were also applicable to evaluate MAM in the primary cultured cells. Therefore, our data indicate that the MAMtrackers are applicable to various cell lines and have a potential for various applications. In addition to their broad applicability, the MAMtracker-based reporters have several advantages over the conventional methods. First, the MAMtrackers are suitable for living cells, unlike evaluations using EM or PLA. Second, the experimental procedures are more straightforward than the conventional methods. Third, the MAMtrackers have advantages in detecting reversible changes in MAM dynamics. Recently, some methods using split GFP have been used in MAM studies.14–16 A split GFP is a powerful tool to visualize and quantify MAM in living cells. However, the irreversible complementation in split GFP resulted in less sensitivity during the recovery from starvation than the MAMtrackers (Figure 4C-E). This difference could be explained by the relatively higher dissociation constant of the MAMtrackers. Therefore, the MAMtrackers are suitable tools to evaluate MAM dynamics in living cells. Mitochondria-ER Length Indicator Nanosensor (MERLIN), another tool for MAM quantification, was also developed recently and is based on bioluminescence resonance energy transfer.36 MERLIN is a similar reporter to MAMtracker-Luc, and they share the same advantages in detecting MAM dynamics. Compared to MERLIN, the MAMtrackers are constructed in a single vector, assuring equal expression levels of the components. This may improve quantification by reducing the variation caused by transfection efficiency.
We used the MAMtrackers to screen for MAM disruption induced by ALS-causative genes and identified most of them compromised MAM integrity. Similar to SOD1, the overexpression of MATR3, VCP, and PFN1 induced MAM disruption in an ALS-linked mutation-specific manner. However, unlike SOD1, the mutant proteins encoded by these genes did not aggregate except for PFNC71G mutant.18 Moreover, unlike SOD1G85R, PFN1C71G did not co-aggregate with MAMtracker-Green. Further investigations are required to elucidate the detailed mechanisms of those ALS-causative gene-mediated MAM disruptions. Overexpression of both wild-type and ALS-linked mutants of TARDBP, TIA1, TAF15, FIG4, CHMP2B, TFG, SQSTM1, TUBA4A, and CHCHD10 decreased the amounts of MAM. Because each of these genes has a wide variety of functions, this implies that MAM is prone to be impaired through the malfunction of various intracellular pathways.
Our result for TARDBP was in accord with a previous report.10 Intriguingly, we found that a TAR DNA-binding protein 43 (TDP-43) mutant devoid of its nuclear localization signal (TDP-43∆NLS) did not alter MAM, suggesting that MAM disruption is rather caused by an excess amount of nuclear TDP-43. Overexpression of TDP-43 in mice shows motor impairment and reduced survival.37, 38 Moreover, our previous study demonstrated that the increased stability of TDP-43 mutants was associated with the accelerated onset of the disease, and TDP-43∆NLS exhibited a substantially shorter half-life in cells.39 Thus, MAM disruption is possibly associated with the gain-of-toxicity of TDP-43 in ALS. Further studies are required to reveal the detailed mechanism.
There is a discrepancy in the effects of FUS and vesicle-associated membrane protein-associated protein B/C (VAPB) on MAM between our study and those of Stoica and colleagues.10, 11 In our study, overexpression of FUS or VAPB resulted in increased or unchanged levels of MAM, respectively. In contrast, Stoica and colleagues reported that overexpression of FUS or VAPB decreased or increased MAM, respectively, and concluded that FUS prevents VAPB-dependent ER–mitochondria tethering to disrupt MAM. The diversity of these phenotypes might be explained by the different cell lines used in these studies; they used NSC-34 cells and mouse spinal cord motor neurons, whereas we used N2a cells. The degree of VAPB-dependent ER–mitochondria tethering might be very low and ignorable in N2a cells. Another possibility is that the MAMtrackers might not be suitable to detect MAM formed by VAPB. Various MAM-tethering proteins have been identified,40suggesting that MAM is a highly heterogeneous microdomain. Therefore, the MAMtrackers may not be localized at VAPB-dependent MAM.
In terms of FUS, we used both the MAMtrackers to demonstrate that wild-type FUS upregulated MAM, suggesting a protective role of FUS in maintaining MAM integrity, at least in N2a cells. Moreover, we found that the FUS-mediated MAM induction requires the nuclear localization of FUS, since FUSP525L, an ALS-linked mutant mislocalized in the cytosol, lost the ability to increase MAM. Consequently, our data support the idea that a loss-of-function mechanism is responsible for FUS-linked ALS.31, 32 Besides, our results suggest that TBK1 also has a novel function that upregulates MAM. This result is reminiscent of TBK1-linked ALS pathology caused by loss-of-function of the gene.33–35 Combined with our previous work on SIGMAR1-mediated maintenance of MAM integrity,3 our data indicate that FUS, TBK1, and SIGMAR1 share a protective role in the maintenance of MAM integrity.
Our study using the MAMtrackers indicated that MAM disruption was a common hallmark in the majority of inherited cases of ALS. MAM serves as functional hubs for various cellular processes and responses,41 including Ca2+ and lipid transfer from the ER to mitochondria, mitochondrial dynamics, ER stress response, autophagy, and inflammation, all of which have been reported as related to ALS pathology. Therefore, although our study is unable to address a causal role of MAM disruption in ALS, it would be reasonable to assume that MAM disruption might be a key pathomechanism in ALS. Remaining questions in this study, regarding the detailed mechanisms of MAM disruption in each inherited ALS gene and the relationship of MAM disruption with sporadic ALS, should also be addressed by further studies.
In this study, we successfully developed two novel MAM reporters, the MAMtrackers, to reveal MAM disruption as a common hallmark in inherited ALS models. The advantages of the MAMtrackers in screening assays would be useful to explore candidate molecules that maintain or compromise MAM integrity. Lastly, the MAMtrackers are applicable to a wide variety of research fields associated with MAM integrity.